Association between Environmental Dioxin-Related Toxicants Exposure and Adverse Pregnancy Outcome: Systematic Review and Meta-Analysis.

Dioxin-related compounds are associated with teratogenic and mutagenic risks in laboratory animals, and result in adverse pregnancy outcomes. However, there were inconsistent results in epidemiology studies. In view of this difference, we conducted a systematic review and meta-analysis to examine this association and to assess the heterogeneity among studies. Comprehensive literature searches were performed to search for relevant articles published in English up to 15 May 2012. In total, we identified 15 studies which included 9 cohort and 6 case control studies. The Cochrane Q test and index of heterogeneity (I(2)) were used to evaluate heterogeneity. In either cohort studies (I(2)=0.89, p<0.0001) or case control studies (I(2)=0.69, p=0.02), significant heterogeneity of risk estimates were observed. Subgroup analyses found no significant increased risk of adverse pregnancy outcome with air dioxin-related compounds exposure (RR=0.99, 95% CI:0.85-1.16), no significant increased risk of spontaneous abortion (SAB) with exposure to food dioxin-related compounds (RR=1.05, 95% CI:0.80-1.37), higher significant risks of low birth weight (LBW) with exposure to food dioxin-related compounds (RR=1.55, 95% CI:1.24-1.94), and higher significant risks of birth defects with maternal solid contaminants dioxin exposure (OR=1.24, 95% CI:1.19-1.29). In conclusion, more evidences are needed to confirm the association between environmental dioxin-related compounds exposure and pregnancy outcome.


Introduction
Many dioxin-related toxicants, including the polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are persistent environmental contaminants. These compounds are characterized by high-affinity binding to the Ah receptor, which are thought to mediate most biological effects by the ligand-Ah receptor complex (1). Dioxin-related toxicants are ubiquitous contaminants of various industrial and combustion processes. They are extremely stable in the environment and have been classified as a type of known human carcinogen (2). In addition to cancer, dioxin-related toxicants may result in other health hazards, such as impaired or altered thyroid hormone regulation (3), immunological functioning (4), and neurological development (5). Furthermore, experimental studies indicate that exposure to tetrachlorodibenzo-p-dioxin (TCDD) is associated with increased teratogenic and mutagenic risks (6), and it has been linked to a variety of adverse pregnancy effects in animals, including spontaneous abortion (SAB) (7) and preterm birth (8). Although reproductive effects of dioxin exposure have been reported in numerous experiments of animals, studies of this association in humans are limited, and the conclusions are always equivocal and often controversial. Some epidemiologic studies have demonstrated that exposure to dioxin-related compounds is associated with higher proportions of adverse outcomes including SAB, stillbirth and preterm delivery, fetal growth restriction, and low birth weight (LBW) (9)(10)(11)(12). However other epidemiologic studies in humans have not shown such effects. Women in the United States who lived near a horse arena that had been sprayed with dioxin-contaminated oil did not have higher rates of fetal or infant mortality, intrauterine growth retardation, LBW, or birth defects compared with unexposed women (13). Studies conducted in a population living in contaminated areas, such as Vietnam veterans, exposed workers, and those affected by chloracne failed to show an association between TCDD exposure and birth defects (14)(15)(16)(17)(18)(19).
The reasons for ambiguous findings in human studies are unknown but likely include the facts that many studies are limited by different methodologies, different endpoints, and small sample size (20). The present study is a meta-analysis relying on published studies in English to explore the relationship between exposure to dioxin-related compounds and adverse pregnancy outcomes. Additionally, our study has focused on general environmental dioxin exposure such as wood preservatives, consuming Swedish east coast fish, food contamination, municipal solid waste incinerator, and chemicals contaminated with TCDD, but not war-related Agent Orange contamination.

Materials and Methods
We followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) criteria (21) for reporting. The data were extracted from published manuscripts, thus no research ethics board approval was necessary.

Data source
Comprehensive literature searches were performed using the PubMed, Springer, Elsevier Digital Dissertations Databases, Scopus, and ISI web of knowledge for relevant articles published in English up to May 2012. Key words used were: "dioxin" or "TCDD" in conjunction with one of the following terms "pregnancy outcome", "reproductive outcomes", "pregnancy loss", "preterm delivery" , "spontaneous abortion", "SAB", "small for gestational age (SGA)", "SGA", "stillbirth", "low birth weight" and "LBW". We extended our search to review the reference list of retrieved articles and performed a manual search as a supplement. Two investigators carefully examined the full texts of the candidate articles to determine whether they met the inclusion criteria for the systematic review and meta-analysis.
The criteria for admitting articles to this study included: i. case-control studies or cohort studies; ii. data culled from studies in humans; iii. maternal or paternal exposure to dioxin-related compounds; and iv. preterm delivery (birth with gestation of less than 37 weeks); SAB (spontaneous loss of an intrauterine pregnancy at less than 20 weeks gestation); and stillbirth (fetal death that occurred at 20 weeks or greater gestation). LBW was defined as birth weight lower than 2500 g.
Case reports, letters, review articles, and abstracts without full texts were excluded from the analysis. Studies of military Agent Orange exposure that occurred during the Vietnam war and adverse pregnancy outcome in military were not considered because exposure of this population were higher than our focus here on environmental levels of dioxin.
When a study had duplicate publications, only the most inclusive publication was considered. For studies with multiple outcomes, only data concerning adverse pregnancy outcome were included in the analysis.

Data extraction
Two investigators carefully and independently extracted the data. In case of inconsistent valuations, agreements were reached following discussion. For each study, the following characteristics were collected: first author, publication year, country, study period, characteristics of study population (exposed and unexposed/cases and controls), exposure definition/data source and measurement, exposed level, case ascertainment, and study results. The relative risks (RRs) or odds ratios (ORs) were extracted and summarized into a 2×2 table format.

Bias assessment among included studies
Risks of biases in the eligible studies were assessed by two authors according to a checklist described by Shah and Balkhair (22). We evaluated the biases according to criteria for sample selection, exposure assessment, outcome assessment, confounder, analytical, and attrition. Different bias risk levels were classified in each category, which included unable to discern, no bias, low bias, moderate bias, and high bias.

Statistical methods
Review Manager 5.0 (http://www.cc-ims.net/ RevMan.) was used for data synthesis and draw forest plots. Heterogeneity assumption between studies was examined by the Q-test and index of inconsistency (I 2 ) (23). A random-effect model using the DerSimonian and Laird method was selected to pool data if there was significant heterogeneity (p<0.05). Otherwise, the fixed-effect model us-ing the Mantel-Haenszel method was conducted. Publication bias was evaluated through the Begg's test, the Egger asymmetry test, and visual inspection of funnel plots by Stata 8.0 (Stata Corporation, College Station, TX, USA). All p values were for the two-sided test and we considered p<0.05 as statistically significant.

Description of studies
This review included 15 studies. Among the 15 studies identified, there were 9 cohort and 6 casecontrol studies. The results of the searches and the articles selection log are reported in figure 1. The characteristics of the 15 studies which included first author, year of publication, country, study period, characteristics of the study population, exposure definition/data source and measurement, exposed level, case ascertainment, and study results are reported in tables 1 and 2.

Quality of included studies
The results of bias assessment of the included studies are shown in table 3. From the 15 studies, 6 had an overall moderate risk of bias and 9 had a low risk of bias. Moderate risk of bias was assigned mostly due to indirect exposure assessment methods used in these studies (22). Various exposure sources and exposure styles were reported on by the different studies, however, the majority exposure components were dioxin or dioxin-like compounds.

Test of heterogeneity
Review Manager 5.0 was used to test the heterogeneity of the 9 cohort and 6 case-control studies. In either the cohort (p<0.0001) or casecontrol (p=0.02) studies, we noted significant heterogeneity of the risk estimates as shown in table 4. The I 2 was 0.89 for cohort studies and 0.69 for case-control studies. Pan et al.

Subgroup analysis
Significant heterogeneities were observed in both cohort and case control studies, which could possibly be attributed to the differences in population under investigation, exposure source, exposed level, and pregnancy outcome. Thus, further subgroup analyses were needed. The heterogeneity tests of subgroups are shown in table 4. As seen in table 4 and figure 2A-D, we found no significant increased risk of adverse pregnancy outcome with exposure to air dioxin-related compounds (RR=0.99, 95% CI:0.85-1.16, Fig 2A). There was no significant increased risk of SAB with food dioxin-related compounds (RR=1.05, 95% CI: 0.80-1.37, Fig 2B). However there was a significantly increased risk noted in LBW to food dioxin-related compounds (RR=1.55; 95% CI: 1.24-1.94, Fig 2C) and in birth defects with maternal exposure to solid contaminants dioxin (OR=1.24; 95% CI:1.19-1.29, Fig 2D).

Sensitivity analyses and publication bias
Sensitivity analyses were conducted to assess whether each individual study affected the final results. These analyses suggested that no individual study affected the results in all subjects using the exclusion method step by step (data not shown).
Funnel plots of all studies revealed no asym-metrical distribution of ORs or RRs (Fig 3 A-C), which suggested no significant publication bias in the overall studies (Egger's test: t=-1.98, p=0.073). When studies were stratified by study design, the funnel plots and Egger's test also indicated no publication bias among either case-control (Egger's test: t=-0.98, p=0.360) or cohort (Egger'

Principal findings
In this systematic review of 15 studies, we identified variable effects of exposure to dioxin-related compounds on adverse pregnancy outcome which included LBW, SAB, SGA, stillbirth, and birth defects. There was an association between exposures to food dioxin-related toxicants with LBW; maternal exposure to solid contaminants dioxin was associated with birth defects. The association between exposure to dioxin-related toxicants and other adverse pregnancy outcomes was inconclusive. Hence, investigation of the effect of dioxin exposure on adverse pregnancy outcomes is challenging, and further studies with improved methodologies are needed to establish or refute an associative relationship.
In 2002, Ngo et al. (37) systematically reviewed studies of parental exposure to Agent Orange, dioxin-contaminated defoliants which used in Vietnam War, which appeared to be associated with an increased risk of birth defects. However, their study had a significant heterogeneity of effects across study populations. Their conclusion was based on 11 Vietnamese cohort and cross-sectional studies and 6 non-Vietnamese cohort studies, which suggested that the subjects had a specific higher-level of dioxin exposure. In their 2008 systematic review of 7 studies about the association between paternal exposure to Agent Orange and spina bifida, Ngo et A B C al. (38) concluded that paternal exposure to Agent Orange was associated with increased risk of spina bifida, however when analyzed according to the study design, the association was not statistically significant for the cohort studies.

Strength and weakness of the review
Epidemiologic studies of the association between exposure to TCDD or related compounds (e.g., other dioxins, furans, and dioxin-like PCBs) and pregnancy outcome in humans are inconsistent -probably due to limitations incurred by inadequate methodology, inappropriate endpoints, and small sample size, among other reasons. However, meta-analyses have the advantages that increase statistical power by pooling the results from small individual studies and also permit examination of the variability between studies (39).
This review also has some limitations. We restricted our searches to English publications due to the scope of information that might not be available in other languages. We did not include gray literature, abstracts, conference articles, and proceedings in this systematic review. The methodology quality of included studies was assessed solely according to the description by Shah and Balkhair (22). However, a number of internal validity or "risk of bias" tools have been developed for observational studies (40)(41)(42)(43) and the Agency for Healthcare Research and Quality has released recent guidance on this topic (44). In addition, none of the included studies assessed impact based on only TCDD toxicity. Research on the health effects of TCDD needs to consider not only TCDD but also other factors such as moisture, temperature and nutrition, etc. (22,45,46). Thus, it is important to take into account these limitations when considering the conclusions of this review.

Potential non-causal explanations
There were likely multiple reasons for the failure to find an association between exposure to dioxinrelated compounds and adverse pregnancy outcomes in humans.
Compared to experimental studies, investigative studies in humans are not like adult animals who are better equipped to combat dioxin exposure. Mocarelli et al. (47) described a permanent reduction in sperm quality in men exposed to TCDD prior to puberty. Numerous studies have demonstrated that males can confer a risk of preterm birth, pre-eclampsia and other adverse pregnancy outcomes to their partners, (48,49) though the mechanisms have not been established. However, animal models clearly demonstrate that early life (in utero) toxicant exposures do have adult reproductive effects (50,51). Thus, there exists the possibility in humans that the timing of exposure is critical, but difficult to assess with regard to subsequent pregnancy outcome.
Second, but equally important, emerging studies indicate that TCDD exposure alters the impact of a subsequent environmental stressor (i.e., infection) (52). Therefore, a TCDD associated adverse outcome in pregnancy may not be noted if the mother is otherwise healthy, but may only become a risk if a secondary stressor is present. Additionally, adverse pregnancy outcome such as LBW, SAB or SGA have varied and multiple etiologies and pathogenesis.
Potential bias such as selection and measurement bias, confounding, and publication bias exist in all meta-analyses, particularly in observational studies.
Because of different exposure states, different exposure sources, commonality of exposure, and the lack of using biomarkers to measure individual exposure, it is possible that some individuals had either minimal or no TCDD exposure level, which might entail further exposure misclassification bias. In some studies, the assessment of exposure was made based on groups of study participants according to their residential location, workplace or intake of TCDD contaminated food history. Some studies used the address at the time delivery to characterize a mother's exposure and did not take into account a mother's residential history, which might also lead to exposure misclassification. Based solely on one single address, the exposure estimated in those studies could only partially reflect an individual's true exposure. Furthermore, the assessment of exposure and outcome in some studies has been made largely through interviewing parent(s) at the time of data collection, which generally occurred 10 years or more after exposure. This approach is known to miss those who died in both exposed and non-exposed groups, and can introduce survival bias. Some other studies identified only certain specific, but not all, adverse pregnancy outcomes which were of interest. These methods of data collection inevitably excluded malformed cases, which were not known by their parents. This type of bias would have underestimated, not overestimated, the risk of dioxin and adverse pregnancy outcome.

Possible mechanism
It is impossible to explain the mechanism of the association between exposure to dioxinrelated toxicants and adverse pregnancy outcomes in this systematic review. However, a large number of previous animal studies indicate that TCDD is associated with a developmental syndrome that involves hydronephrosis, cleft palate, and fetal thymic atrophy in mice (53), and increased fetal loss and reduction in birth weight in experimental studies in rodents and monkeys (7,(54)(55)(56)(57)(58). Meanwhile, large experimental studies demonstrate that the toxic effects of TCDD are mediated by aryl hydrocarbon receptor (AhR). TCDD is the most potent activator of AhR. The activated AhR has been described to cause toxic effects reminding symptoms of vitamin A depletion such as respiratory tract and bile duct keratinization, dermal and epithelial lesions, thymus atrophy, immunodeficiency or impaired reproduction (59,60). Some studies suggest that there are interactions between AhR ligands and the retinoid transport system, metabolism and signaling because it has been described that at least some of the negative effects caused by AhR ligands in TCDD exposed animals can be compensated by supplementation with vitamin A (59,61). We speculate that the reason of intaking TCDD contaminated fish and Yusho oil had no association with SAB maybe partly because fish and oil are rich in vitamin A.

Conclusion
The association between exposure to environmental dioxin-related toxicants, with the exception of Agent Orange, and pregnancy outcome is inconclusive. Thus, examination of dioxin exposure and pregnancy outcome is challenging. The biological mechanism of this association and methodological limitations of the studies warrant the consideration of conducting large-scale, well-designed studies in the future. Future studies need to include biologi-cal measures of exposure.